In this application we will use the interferon nomenclature announced in Nature, 286, p. 110 (July 10, 1980). "IFN" will designate interferon and "IFN-.gamma." will designate gamma interferon, formerly known as "immune" interferon.
Two classes of interferon ("IFN") are known to exist. Interferons of type I are small, acid stable proteins that render cells resistant to viral infection (A. Isaacs and J. Lindenmann, "Virus Interference I. The Interferon", Proc. Royal Soc. Ser. B., 147, pp. 258-67 (1957); W. E. Stewart II, The Interferon System, Springer-Verlag (2 ed.) (1981) (hereinafter "The Interferon System")). Type II IFNs are acid labile. At present, they are poorly characterized. Although rather cell specific (The Interferon System, pp. 135-45), IFNs are not virus specific. IFNs protect cells against a wide spectrum of viruses.
Type II interferons can be produced spontaneously, or in response to various inducers, such as viruses, microorganisms, mitogens, viral or bacterial antigens, or in response to antibiotics, endotoxins or other microbial products (The Interferon System, pp. 148-49). Most usually, mitogens such as Staphylococcal enterotoxin, galactose oxidase, phytohemagglutinin, concanavalin, Corynebacterium parvum and mixed lymphocyte cultures are used. Type II interferons have also been reported to be produced from a variety of sources; for example, cerebrospinal fluid in CNS disease, leukocytes induced with phytohemagglutinin, lymphocytes induced with antilymphocyte serum, lymphocytes induced with macrophages and mitogens, tonsil lymphocytes induced with mitogens, herpes-sensitized lymphocytes induced with macrophages and herpes virus antigen, rubella-virus sensitized lymphocytes induced with rubella virus, lymphoblastoid cells, bone marrow of multiple myeloma patients, lymphocytes from a leukemic T-cell line and lymphocytes isolated from buffy coats, human milk or by plasmaphoresis (The Interferon System, pp. 146-47; I. Nathan et al., "Immune (.gamma.) Interferon Produced By A Human T-Lymphoblast Cell Line", Nature, 292, pp. 842-43 (1981); M. A. Heller et al., "Lymphokine Production By Human Milk Lymphocytes", Infection And Immunity, 32, pp. 632-36 (1981); G. R. Klimpel et al., "Differential Production Of Interferon And Lymphotoxin By Human Tonsil Lymphocytes", Cellular Immunity, 20, pp. 187-96 (1975); M. DeLey et al., "Interferon Induced In Human Leucocytes By Mitogens: Production, Partial Purification And Characterization", Eur. J. Immunol., 10, pp. 877-83 (1980); M. Wiranowska-Stewart and W. E. Stewart II, "Determination Of Human Leukocyte Population Involved In Production Of Interferon Alpha And Gamma", J. Interferon Research, 1, pp. 233-44 (1981)). Considering this heterogeneity, Type II IFN production upon mitogenic stimulation appears to depend upon the source of the lymphocytes and upon the isolation procedure employed.
Interferon therapy against viruses and tumors or cancers has been conducted at varying dosage regimes and under several modes of administration (The Interferon System, pp. 306-22). For example, interferon has been effectively administered orally, by inoculation--intravenously, intramuscularly, intranasally, intradermally and subcutaneously--and in the form of eye drops, ointments and sprays. It is usually administered one to three times daily in dosages of 10.sup.4 to 10.sup.8 units. The extent of the therapy depends on the patient and the condition being treated. For example, virus infections are usually treated by daily or twice daily doses over several days to two weeks and tumors and cancers are usually treated by daily or multiple daily doses over several months or years. The most effective therapy for a given patient must of course be determined by the attending physician, who will consider such well known factors as the course of the disease, previous therapy, and the patient's response to interferon in selecting a mode of administration and a dosage regime.
As an antiviral agent, human interferon ("HuIFN") has been used to treat the following: respiratory infections (Texas Reports, pp. 486-96); herpes simplex keratitis (Texas Reports, pp. 497-500; R. Sundmacher, "Exogenous Interferon In Eye Diseases", International Virology IV, The Hague, Abstract nr. w2/11, p. 99 (1978)); acute hemorrhagic conjunctivitis (Texas Reports, pp. 501-10); adenovirus keratoconjunctivitis (A. Romano et al., ISM Memo I-A8131 (October, 1979)); varicella zoster (Texas Reports, pp. 511-15); cytomegalovirus infection (Texas Reports, pp. 523-27); and hepatitis B (Texas Reports, pp. 516-22). See also The Interferon System, pp. 307-21. However, large-scale use of IFN as an antiviral agent requires larger amounts of IFN than heretofore have been available.
IFN has other effects in addition to its antiviral action. For example, it antagonizes the effect of colony stimulating factor, inhibits the growth of hemopoietic colony-forming cells and interferes with the normal differentiation of granulocyte and macrophage precursors (Texas Reports, pp. 343-49). It also inhibits erythroid differentiation in DMSO-treated Friend leukemia cells (Texas Reports, pp. 420-28).
IFN may also play a role in regulation of the immune response. For example, depending upon the dose and time of application in relation to antigen, IFN can be both immunopotentiating and immunosuppressive in vivo and in vitro (Texas Reports, pp. 357-69). In addition, specifically sensitized lymphocytes have been observed to produce IFN after contact with antigen. Such antigen-induced IFN could therefore be a regulator of the immune response, affecting both circulating antigen levels and expression of cellular immunity (Texas Reports, pp. 370-74). IFN is also known to enhance the activity of killer lymphocytes and antibody-dependent cell-mediated cytotoxicity (R. R. Herberman et al., "Augmentation By Interferon Of Human Natural And Antibody-Dependent Cell-Mediated Cytotoxicity", Nature, 227, pp. 221-23 (1979); P. Beverley and D. Knight, "Killing Comes Naturally", Nature, 278, pp. 119-20 (1979); Texas Reports, pp. 375-80; J. R. Huddlestone et al., "Induction And Kinetics Of Natural Killer Cells In Humans Following Interferon Therapy", Nature, 282, pp. 417-19 (1979); S. Einhorn et al., "Interferon And Spontaneous Cytotoxicity In Man. II. Studies In Patients Receiving Exogenous Leukocyte Interferon", Acta Med. Scand. 478-83 (1978)).
Killer lymphocytes and antibody-dependent cell-mediated cytotoxicity may be directly or indirectly involved in the immunological attack on tumor cells. Therefore, in addition to its use as an antiviral agent, IFN has potential application in antitumor and anticancer therapy and in immunomodulation agents and methods (The Interferon System, pp. 319-21, 394-99). It is now known that IFNs affect the growth of many classes of tumors in many animals (The Interferon System, pp. 292-304). Interferons, like other antitumor agents, seem most effective when directed against small tumors. The antitumor effects of animal IFN are dependent on dosage and time, but have been demonstrated at concentrations below toxic levels. Accordingly, numerous investigations and clinical trials have been and continue to be conducted into the antitumor and anticancer properties of HuIFNs. These include treatment of several malignant diseases such as osteosarcoma, acute myeloid leukemia, multiple myeloma and Hodgkin's disease (Texas Reports, pp. 429-35). Although the results of these clinical tests are encouraging, the antitumor, anticancer and immunomodulation applications of IFNs have been severely hampered by lack of an adequate supply of purified IFN.
At the biochemical level, IFNs induce the formation of at least three proteins: a protein kinase (B. Lebleu et al., "Interferon, Double-Stranded RNA And Protein Phosphorylation", Proc. Natl. Acad. Sci. USA, 73, pp. 3107-11 (1976); A. G. Hovanessian and I. M. Kerr, "The (2'-5') Oligoadenylate (ppp A2'-5A2'-5'A) Synthetase And Protein Kinase(s) From Interferon-Treated Cells", Eur. J. Biochem., 93, pp. 515-26 (1979)), a (2'-5')oligo(A) synthetase (A. G. Hovanessian et al., "Synthesis Of Low-Molecular Weight Inhibitor Of Protein Synthesis With Enzyme From Interferon-Treated Cells", Nature, 268, pp 537-39 (1977); A. G. Hovanessian and I. M. Kerr, Eur. J. Biochem., supra) and a phosphodiesterase (A. Schmidt et al., "An Interferon-Induced Phosphodiesterase Degrading (2'-5') Oligoisoadenylate And The C-C-A Terminus Of tRNA", Proc. Natl. Acad. Sci. USA, 76, pp. 4788-92 (1979)).
Interferons have been classified into three groups--.alpha., .beta. and .gamma.. Of these, .alpha. and .beta. interferons are acid stable, type I interferons, while .gamma.-interferon is an acid labile, type II interferon. In addition, the three IFNs are antigenically distinct from each other.
IFN-.alpha. and IFN-.beta. are better characterized than IFN-.gamma.. IFN-.gamma. is reported to be a glycoprotein (A. Mizrami et al., "Glycosylation Of Interferon", J. Biol. Chem., 253, pp. 7612-15 (1978); M. P. Langford et al., "Large-Scale Production And Physicochemical Characterization Of Human Immune Interferon", Infection And Immunity, 26, pp. 36-41 (1979); The Interferon System, pp. 107-08). It has also been reported to have a molecular weight of 40,000-46,000 daltons, with the possibility that its glycosylated form has a molecular weight of 65,000-70,000 daltons. In addition to being acid labile (at pH 2), IFN-.gamma. has been reported to be inactivated after 1 h at 56.degree. C. See also DeLey et al., supra; Y. K. Yip et al., "Partial Purification And Characterization Of Human .gamma. (Immune) Interferon", Proc. Natl. Acad. Sci. USA, 78, pp. 1601-605 (1981); M. P. Langford et al., "Large-Scale Production And Physicochemical Characterization Of Human Immune Interferon", Infection And Immunity, 26, pp. 36-41 (1979). And, it has been reported that IFN-.gamma. recognizes a different cell receptor than IFN-.alpha. or IFN-.beta. (A. A. Branca and C. Baglioni, "Evidence That Types I And II Interferons Have Different Receptors", Nature, 294, pp. 768-70 (1981)).
In addition to its antiviral activity, IFN-.gamma. is reported to display antitumor activity. Moreover, as compared to IFN-.alpha. and IFN-.beta., IFN-.gamma.'s antitumor activity seems, at least in mice, to result in tumor regression. In addition, its activation of natural killer cells does not reach a plateau as observed for IFN-.alpha. and IFN-.beta. and IFN-.gamma. appears to be less inhibited by circulating levels of gangliosides than are IFN-.alpha. and IFN-.beta. (H. Ankel et al., "Mouse Fibroblast (Type I) And Immune (Type II) Interferons: Pronounced Differences In Affinity For Gangliosides And In Antiviral And Antigrowth Effects On Mouse Leukemia L-1210R Cells", Proc. Natl. Acad. Sci. USA, 77, pp. 2528-32 (1980)). Therefore, it appears that cells or tumors that display a poor response to IFN-.alpha. or IFN-.beta. may be effectively treated with IFN-.gamma. (e.g., Crane et al., J. Natl. Cancer Institute, 61, p. 891 (1978); Barn et al., Abstract N.Y. Acad. Sci., No. 11 (Oct. 23-26, 1979); Blalock et al., Cellular Immunology, 49, pp. 390-94 (1980); B. Y. Rubin and S. L. Gupta, "Differential Efficacies Of Human Type I And Type II Interferons As Antiviral And Antiproliferative Agents", Proc. Natl. Acad. Sci. USA, 77, pp. 5928-32 (1980)).
Another use of IFN-.gamma. may be as an immunoregulatory agent. IFN-.gamma. has been found to be useful in treating rheumatoid arthritis and in the treatment of certain allergies. IFN-.gamma. is also useful in combination with other compounds. For example, when combined with tumor necrosis factor ("TNF") tumor growth is inhibited beyond that seen with IFN-.gamma. or TNF alone (see European patent application 131,789). IFN-.gamma. can also be combined with other lymphokines, such as IFN-.alpha., IFN-.beta. and Interleukin 2, or with anti-inflammatories for enhanced effect.
HuIFN-.gamma., like many human proteins, may also be polymorphic. Therefore, cells of particular individuals may produce IFN-.gamma. species within the more general IFN-.gamma. class which are physiologically similar but structurally slightly different from the prototype of the class of which it is a part. Therefore, while the protein structure of the IFN-.gamma. may be generally well-defined, particular individuals may produce IFN-.gamma.'s that are slight variations thereof.
Both a family of HuIFN-.alpha.'s and a HuIFN-.beta. have been produced in appropriate hosts using recombinant DNA technology (e.g., S. Nagata et al., "Synthesis In E. coli Of A Polypeptide With Human Leukocyte Interferon Activity", Nature, 284, pp. 316-20 (1980) (IFN-.alpha.) and R. Derynck et al., "Expression Of The Human Fibroblast Interferon Gene In Escherichia coli", Nature, 287, pp. 193-202 (1980) (IFN-.beta.)). Such techniques have allowed further characterization and initial clinical trials of the HuIFN-.alpha. and HuIFN-.beta..
Today, the extremely small quantities of HuIFN-.gamma. that are available are produced by a human cell line grown in tissue culture or more usually from leukocytes collected from blood samples. These processes are low yield and expensive ones. They have not provided sufficient HuIFN-.gamma. to permit further characterization or clinical trials to elucidate further the antiviral, antitumor or immunomodulation activities of IFN-.gamma. or to compare those activities to the antiviral, antitumor and immunomodulation activities of IFN-.alpha. and IFN-.gamma..